Controlling biofilms of gram-positive pathogenic bacteria.

Many bacteria can form aggregates on interfaces, called biofilms, where they are much more protected against toxic agents such as antibiotics or antibodies. Bacteria organized in biofilms are therefore very difficult to control and often even high dosages of antibiotics cannot clear infectious biofilms. To form biofilms bacteria have to start a complex genetic program to switch from planktonic to sessile lifestyle.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. II. Dynamic response to famine and feast, activation of the methylglyoxal pathway and oscillatory behaviour.

The metabolic dynamics of the Escherichia coli K-12 strain TG1 to feast and famine were studied in glucose-limited steady-state cultures by up- and downshifts of the dilution rate, respectively. An uncoupling of anabolic and catabolic rates was observed upon dilution rate upshifts, apparent through immediately increased glucose uptake rates which were not accompanied by an immediate increase of the growth rate but instead resulted in the temporary excretion of methylglyoxal, D- and L-lactate, pyruvate and, after a delay, acetate. The energetic state of the cell during the transient was followed by measuring the adenylate energy charge, which increased within 2 min after the upshift and declined thereafter until a new steady-state level was reached.

Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state.

The Escherichia coli K-12 strain TG1 was grown at 28 degrees C in aerobic glucose-limited continuous cultures at dilution rates ranging from 0.044 to 0.415 h(-1).

Role of host genetic factors in susceptibility to group A streptococcal infections.

Background & Objectives: Epidemiological evidences indicate that host genetic factors might be critical in determining susceptibility to infection with group A streptococci (GAS). The objective of the present study was to determine the extent to which the genetic background of the mouse strain affected induction and resolution of GAS infection.

Methods: Several inbred mouse strains were intravenously infected with Streptococcus pyogenes strain A20 and the mean survival times of mice was recorded overtime.

Characterization of the interaction of the pneumococcal surface protein SpsA with the human polymeric immunoglobulin receptor (hpIgR).

Background & Objectives: The polymeric immunoglobulin receptor (pIgR) is produced by mucosal epithelial cells and plays a crucial role in mucosal immunity. At the basolateral surface of mucosal cells, the pIgR binds predominantly polymeric immunoglobulins, such as dimeric IgA and polymeric IgA (pIgA) and mediates their transport across the polarized cells. This results in apical release of secretory component (SC), either free or bound covalently to IgA, forming secretory IgA (SIgA).

Selective leakage of host-cell proteins during high-cell-density cultivation of recombinant and non-recombinant Escherichia coli.

Significant leakage of host-cell proteins into the culture medium occurred during high-cell-density cultivation of E. coli. Identification of these medium proteins revealed almost exclusively a periplasmic origin.

Thalassolituus oleivorans gen. nov., sp. nov., a novel marine bacterium that obligately utilizes hydrocarbons.

An aerobic, heterotrophic, Gram-negative, curved bacterial strain, designated MIL-1T, was isolated by extinction dilution from an n-tetradecane enrichment culture that was established from sea water/sediment samples collected in the harbour of Milazzo, Italy. In the primary enrichment, the isolate formed creamy-white, medium-sized colonies on the surface of the agar. The isolate did not grow in the absence of NaCl; growth was optimal at 2.

A dual lethal system to enhance containment of recombinant micro-organisms.

Active containment systems based on the controlled expression of a lethal gene are designed to increase containment of recombinant micro-organisms used for environmental applications. A major drawback in containment is the existence of mutations that generate surviving cells that cease to respond to the toxic effect of the lethal function. In this work the authors have developed for the first time a strategy to reduce the problem of mutations and increase the efficiency of containment based on the combination of two lethal functions acting on different cellular targets of major concern in containment, DNA and RNA, and whose expression is under control of different regulatory signals.

Degradation of alkanes and highly chlorinated benzenes, and production of biosurfactants, by a psychrophilic Rhodococcus sp. and genetic characterization of its chlorobenzene dioxygenase.

Rhodococcus sp. strain MS11 was isolated from a mixed culture. It displays a diverse range of metabolic capabilities.

Thermoleophilum album and Thermoleophilum minutum are culturable representatives of group 2 of the Rubrobacteridae (Actinobacteria).

Analysis of the morphological and genotypic properties of three obligately thermophilic strains of Thermoleophilum album and Thermoleophilum minutum, originally described as green non-sulfur bacteria, indicates that these taxa belong to the Rubrobacter subdivision of the Actinobacteria. EM of the cell wall clearly showed morphology typical of Gram-positive bacteria. A comparison of 16S rRNA gene sequences, including signature nucleotide pairs and secondary structural features considered diagnostic for the subclass Rubrobacteridae, revealed that the three strains of Thermoleophilum were highly similar and should be considered as members of group 2 of this subclass, represented up to now only by uncultured organisms.

Induction of NF-kappaB nuclear translocation in human respiratory epithelial cells by group A streptococci.

The activation of the transcription factor NF-kappaB and the production of inflammatory mediators play an essential role in the host response to pathogenic organisms. The objective of this study was to investigate the ability of group A streptococci (GAS) to stimulate the nuclear translocation of NF-kappaB in cultured human epithelial (HEp-2) cells. Infection of HEp-2 cells with a strain of Streptococcus pyogenes capable to efficiently internalize HEp-2 cells (strain A40) resulted in translocation of NF-kappaB during the first 15 min of infection, reaching a peak after 30 min that persisted at slightly lower levels 1h thereafter.

Phagocytosis of bacille Calmette-Guérin-infected necrotic macrophages induces a maturation phenotype and evokes antigen-presentation functions in dendritic cells.

The interaction of pathogens with dendritic cells (DCs) seems to play a critical role in the initiation of the immune response. Tissue damage and induction of an inflammatory reaction are events frequently associated with the progression of the infection. Although DCs are very efficient at phagocytosing pathogens, the capacity of these cells to uptake microbes from a necrotic environment has not yet been proven.

Influence of proteins Bsp and FemH on cell shape and peptidoglycan composition in group B streptococcus.

Group B streptococcus (GBS) is surrounded by a capsule. However, little is known about peptidoglycan metabolism in these bacteria. In the present study, a 65 kDa protein was isolated from the culture supernatant of GBS and N-terminally sequenced, permitting isolation of the corresponding gene, termed bsp.

Multiphasic kinetics of transformation of 1,2,4-trichlorobenzene at nano- and micromolar concentrations by Burkholderia sp. strain PS14.

The transformation of 1,2,4-trichlorobenzene (1,2,4-TCB) at initial concentrations in nano- and micromolar ranges was studied in batch experiments with Burkholderia sp. strain PS14. 1,2,4-TCB was metabolized from nano- and micromolar concentrations to below its detection limit of 0.

Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans.

We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates. Neither of the organisms was able to grow with chlorodibenzofurans alone. Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule.

Combined use of 16S ribosomal DNA and 16S rRNA to study the bacterial community of polychlorinated biphenyl-polluted soil.

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries.

An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics.

A systematic evaluation of the value and potential of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure has been undertaken. The reproducibility and robustness of the method has been assessed using environmental DNA samples isolated directly from PCB-polluted or pristine soil, and subsequent polymerase chain reaction (PCR) amplification of total community 16S rDNA. An initial investigation to assess the variability both within and between different polyacrylamide gel electrophoresis (PAGE) runs showed that almost identical community profiles were consistently produced from the same sample.

'Clay hutches': a novel interaction between bacteria and clay minerals.

Biofilm formation on a low-energy substratum floating on the surface of a water column overlying a polychlorinated biphenyl (PCB)-contaminated sandy clay soil was followed by light and electron microscopy. The biofilms that developed consisted of a dense lawn of clay aggregates, each one of which contained one or more bacteria, phyllosilicates and grains of iron oxide material, all held together by bacterial extracellular polysaccharides (EPS). The clay leaflets were arranged in the form of 'houses of cards' and gave the aggregates the appearance of 'hutches' housing the bacteria.

Identification of the metabolically active members of a bacterial community in a polychlorinated biphenyl-polluted moorland soil.

The presumptive metabolically active members of a bacterial community in a moorland soil in Germany, highly polluted with polychlorinated biphenyls (PCBs), were identified by sequencing of cloned reverse transcription-polymerase chain reaction (RT-PCR) amplification products of 16S rRNA generated from total RNA extracts. Analysis of the 16S rRNA clone library revealed a considerable diversity of metabolically active bacteria in the soil, despite the acidic pH and high concentrations of PCBs. Cloned sequence types clustered within the Proteobacteria (34% alpha-, 33% beta- and 7% gamma-subclasses), the Holophaga-Acidobacterium phylum (14%), the Actinobacteria (6.

Towards elucidation of microbial community metabolic pathways: unravelling the network of carbon sharing in a pollutant-degrading bacterial consortium by immunocapture and isotopic ratio mass spectrometry.

Although much information on metabolic pathways within individual organisms is available, little is known about the pathways operating in natural communities in which extensive sharing of nutritional resources is the rule. In order to analyse such a consortium pathway, we have investigated the flow of 4-chlorosalicylate as carbon substrate within a simple chemostat microbial community using 13C-labelled metabolites and isotopic ratio mass spectrometric analysis of label enrichment in immunocaptured member populations of the community. A complex pathway network of carbon sharing was thereby revealed, involving two different metabolic routes, one of which is completely novel and involves the toxic metabolite protoanemonin.

Co-operative binding of human fibronectin to Sfbl protein triggers streptococcal invasion into respiratory epithelial cells.

Streptococcal fibronectin binding protein I (SfbI) mediates adherence to and invasion of Streptococcus pyogenes into human epithelial cells. In this study, we analysed the binding activity of distinct domains of SfbI protein towards its ligand, the extracellular matrix component fibronectin, as well as the biological implication of the binding events during the infection process. By using purified recombinant SfbI derivatives as well as in vivo expressed SfbI domains on the surface of heterologous organism Streptococcus gordonii, we were able to dissociate the two major streptococcal target domains on the human fibronectin molecule.

Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria.

A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA. The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE).

Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter.

We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette. Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental. In addition to their suitable cloning features (e.

Characterization of SepL of enterohemorrhagic Escherichia coli.

The sepL gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the sepL gene of the enterohemorrhagic Escherichia coli (EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that sepL is transcribed monocistronically and independently from the esp operon located downstream, which codes for the secreted proteins EspA, -D, and -B.